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Comparative Development of Intertracheary Pit Membranes in Abies Firma and Metasequoia Glyptostroboides

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Comparative Development of Intertracheary Pit Membranes in Abies Firma and Metasequoia Glyptostroboides IAWA Journal, Vol. 29 (3), 2008: 277–289 COMPARATIVE DEVELOPMENT OF INTERTRACHEARY PIT MEMBRANES IN ABIES FIRMA AND METASEQUOIA GLYPTOSTROBOIDES Roland Dute, LaToya Hagler and Adam Black Department of Biological Sciences, Auburn University, Life Sciences Building, Auburn, Alabama 36849-5407, U.S.A. SUMMARY This study compares intertracheary pit membrane structure and ontogeny in Abies firma (Pinaceae) and Metasequoia glyptostroboides (Cupres- saceae). Initial phases of pit membrane development are the same for both species. Branched plasmodesmata are present in the earliest stages of pit membrane development observed. Torus thickening of the pit membrane occurs early in pit development prior to pit border initiation. During pit border enlargement, plastids frequently occlude the apertures. Cell lysis is associated with complete wall matrix removal from pit membranes of Metasequoia. By contrast, cell lysis in Abies results in loss of matrix ma- terial from the margo, whereas the torus remains largely unaffected. Torus extensions in pit membranes of A. firmaretain variable amounts of matrix material. Either a difference in chemical composition of the torus or a difference in autolytic enzymes is hypothesized to explain developmental differences between pit membranes of the two species. Key words: Abies, Metasequoia, pit membrane, plasmodesma, plastid, torus, torus extension. INTRODUCTION Recent physiological work has shown the importance of torus-margo pitting to efficient water flow in tracheids of conifers (Hacke et al. 2004; Pittermann et al. 2005). At the same time the ability of the impermeable torus to prevent air seeding is well known (Zim- mermann 1983; Hacke et al. 2004). Earlier studies of mature pit membranes in gymno- sperms have shown the diversity of torus structure (Bauch et al. 1972). In particular, the amount of matrix material remaining within a mature torus can range from considerable (e.g. Abies – Parham & Baird 1973; Sano et al. 1999) to none (Ginkgo – Dute 1994). Metasequoia is another genus in which matrix loss from the torus occurs during cell maturation (personal observation). It was decided to compare pit membrane develop- ment in Abies firma(Pinaceae) and Metasequoia glyptostroboides (Cupressaceae). One question involves time of torus initiation. Torus development in conifers is thought to occur during the primary wall stage of growth (Bauch et al. 1968). In agreement with these findings, Parham and Baird (1973) found torus ontogeny in Abies balsamea largely completed by the beginning of pit border formation. However, Timell (1979) indicates that for this same species torus formation begins at the same time as secondary wall deposition. No information exists for time of torus initiation Downloaded from Brill.com10/06/2021 11:20:42PM via free access 278 IAWA Journal, Vol. 29 (3), 2008 Dute, Hagler & Black — Pit membranes of Abies and Metasequoia 279 in Metasequoia. In eudicotyledons, time of initiation correlates with method of torus manufacture as well as with torus chemistry (Coleman et al. 2004). A second set of questions involves tori and plasmodesmata. Plasmodesmata are com- mon in intertracheary pit membranes of conifers and Ginkgo (q.v. discussion in Dute 1994). Various authors have shown that tori of Abies have plasmodesmata (Timell 1979; Sano et al. 1999). Although no such work has been done on Metasequoia, in Ginkgo, whose torus also loses its matrix material, plasmodesmata disappear at maturity (Dute 1994). Therefore, in the present study, particular attention was paid to plasmodesmata – their time of formation, structure, and final disposition. Finally, torus extensions are well-known from the pit membranes of various species of Abies (Bauch et al. 1972; Imamura et al. 1974; Sano et al. 1999, and literature cited therein). We decided to characterize the anatomy of these structures in Abies firma using SEM and TEM. MATERIALS AND METHODS Branch specimens of 2–4 mm diameter were collected from two individuals each of Abies firma Siebold et Zucc. and Metasequoia glyptostroboides Miki ex Hu & Cheng growing in the Donald E. Davis Arboretum on the Auburn University campus. Five collections were made during the spring of 2005; two collections were made during the spring of 2006. Prior investigations of macerated wood showed average length of branch tracheids to be 1047 μm for Metasequoia and 931 μm for Abies (based on 25 counts each). In order to minimize physical damage to immature tracheids during col- lection and preservation, the following procedure was adopted. Branch segments of approximately 6 mm length were cut and split in half. The periderm was gently scraped away. All cutting and scraping were done in fixative. The specimens were preserved in one-half strength Karnovskyʼs fixative (Karnovsky 1965) in 0.1 M sodium phosphate buffer (pH 7.0) under vacuum at room temperature for one hour. Afterward, the specimen strips were cut to lengths of 2–2.5 mm by removing tissue from both ends, and were cut longitudinally a number of times to make wedges. The wedges were placed in fresh fixative and evacuated for a further hour. The specimens then were kept at 4 °C for five hours. After repeated buffer washes, the specimens were placed in 1% buffered OsO4 for 4.5 hours at room temperature. Another series of buffer washes followed. Specimens then were dehydrated in a cold ethanol series followed by infiltration with acetone. Dehydration was succeeded by infiltration in increasing concentrations of Spurrʼs resin (Spurr 1969) in acetone. After 24 hours in pure resin the tissue was embedded in flat molds at 63 °C for 24 hours. Embedded material was mounted and sectioned for light and transmission electron microscopy (TEM) using a Sorvall MT-2b ultramicrotome. For light microscopy, 2 μm thick sections were heat-fixed to glass slides and stained with 0.5% buffered toluidine blue O. The sectioned material was viewed and photographed with a Nikon Biophot and an attached Nikon D70 camera. For TEM, sections of about 80 nm thickness were cut, placed on copper grids, and stained with uranyl acetate and lead citrate. The sec- tions were viewed with a Zeiss EM10CR using 60 kV accelerating voltage. Downloaded from Brill.com10/06/2021 11:20:42PM via free access 278 IAWA Journal, Vol. 29 (3), 2008 Dute, Hagler & Black — Pit membranes of Abies and Metasequoia 279 For scanning electron microscopy (SEM) branches were split radially and either air- dried or preserved in FAA (formalin-acetic acid-alcohol). The latter specimens were then dehydrated through absolute ethanol, soaked in two changes of HMDS (hexamethyl- disilazane) (Nation 1983) overnight, and allowed to air dry. All dried specimens were mounted on aluminum stubs using carbon-impregnated tape and coated with gold-palla- dium using an Electron Microscopy Sciences 550X sputter coating device. The resulting preparations were viewed with a Zeiss DSM 940 at 15 kV accelerating voltage. Observations from light, SEM, and TEM were predominately from the earlywood. RESULTS Torus initiation and ontogeny With the light microscope, well-developed tori associated with developing pit borders are frequently encountered in immature xylem of both Metasequoia and Abies (Fig. 1 & 2), indicating that tori are initiated early in cell ontogeny. In fact, exceptional views Abbreviations used in this study: D = dictyosomes; E = endoplasmic reticulum; M = mitochon- drion; MA = margo; P = plastid; PH = secondary phloem; PL = plasmodesma; T = torus; TE = torus extension; V = vesicles; VC = vascular cambium; W = warts; X = secondary xylem. Figures 1–4. Initiation of torus. – 1: Light micrograph (LM) of active cambial region in Metase- quoia. – 2: LM of active cambial region in Abies. – 3: TEM of newly initiated torus thickening in Metasequoia. – 4: TEM of newly initiated torus thickening in Abies. Note branched plasmodes- mata. Unlabeled arrows indicate the beginnings of a pit border. — Scale bars = 20 μm for Fig. 1 & 2; 1 μm for Fig. 3 & 4. Downloaded from Brill.com10/06/2021 11:20:42PM via free access 280 IAWA Journal, Vol. 29 (3), 2008 Dute, Hagler & Black — Pit membranes of Abies and Metasequoia 281 Figures 5–7. Cytoplasmic organelles associated with young pit membranes. – 5: Metasequoia. – 6: Metase- quoia. – 7: Abies. Unlabeled arrows in Fig. 5 & 6 de- note cell membrane invaginations enveloping wall mate- rial. — Scale bars = 1 μm for all. using TEM indicate that tori are initiated and can show considerable thickening by the time pit border initiation occurs (Fig. 3 & 4). Figure 4 is particularly instructive. One cell (A) is slightly older than its neighbor (B), and the former shows not only a thicker torus pad, but also initiation of the pit border (unlabeled arrows). Before and during pit border construction the torus is spatially associated with a number of cytoplasmic components, including: plastids, mitochondria, rough ER, dictyosomes, and vesicles (Fig. 5–7). More will be said of plastids later. A microtubule plexus is not associated with torus thickening. Downloaded from Brill.com10/06/2021 11:20:42PM via free access 280 IAWA Journal, Vol. 29 (3), 2008 Dute, Hagler & Black — Pit membranes of Abies and Metasequoia 281 Figures 8–12. Matrix removal from pit mem- branes of Metasequoia. – 8: LM showing pit membrane pre- (A) and postautolysis (B). – 9: TEM of postautolysis pit membrane (sec- tional view). – 10: TEM of torus in the act of losing its matrix material (arrows). – 11: LM of pit membrane in which some matrix material (arrow) still remains in the torus. – 12: SEM of unaspirated pit membrane in sur- face view. — Scale bar = 20 μm for Fig. 8; 1 μm for Fig. 9–11; 2.5 μm for Fig. 12. Cell autolysis follows completion of the pit border. In Metasequoia, the entire pit membrane, including the torus, loses its matrix material (Fig. 8 & 9). With TEM, both torus and margo are distinct, but their thicknesses have decreased from the pre-autolysis stage (Fig. 9). It is possible to find instances in which matrix removal is occurring.
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